ab Adenosylhomocysteinase (AHCY) Inhibitor Screening Assay Kit (Fluorometric)

Transcription

1 + ab Adenosylhomocysteinase (AHCY) Inhibitor Screening Assay Kit (Fluorometric) Instructions for Use For rapid, sensitive and accurate screening of potential Adenosylhomocysteinase (AHCY) inhibitors. This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 23 July 2015

2 Table of Contents INTRODUCTION 1. BACKGROUND 2 2. ASSAY SUMMARY 3 GENERAL INFORMATION 3. PRECAUTIONS 5 4. STORAGE AND STABILITY 5 5. LIMITATIONS 5 6. MATERIALS SUPPLIED 6 7. MATERIALS REQUIRED, NOT SUPPLIED 6 8. TECHNICAL HINTS 7 ASSAY PREPARATION 9. REAGENT PREPARATION SAMPLE PREPARATION 10 ASSAY PROCEDURE and DETECTION 11. ASSAY PROCEDURE and DETECTION 11 DATA ANALYSIS 12. CALCULATIONS TYPICAL DATA 15 RESOURCES 14. QUICK ASSAY PROCEDURE TROUBLESHOOTING FAQ 19 Discover more at 1

3 INTRODUCTION 1. BACKGROUND Adenosylhomocysteinase (AHCY) Inhibitor Screning Kit (Fluorometric) (ab204694) is a simple assay to study, screen or characterize potential inhibitors of AHCY. In this assay, the homocysteine generated from the breakdown of S-Adenosyl Homocysteine (SAH) is measured using a Thiol Detecting Reagent which results in enhanced fluorescence that can be measured at Ex/Em = 392/482 nm. The AHCY reaction is reversible, and therefore Adenosine Deaminase is included in the reaction to ensure that the reaction proceeds towards hydrolysis of SAH. In the presence of AHCY inhibitor, there is a decrease in fluorescence of the Thiol Detecting Reagent. Adenosine Deaminase S-Adenosyl Homocysteine + AHCY Homocysteine + Adenosine Inosine Homocysteine + Thiol Detecting Probe Enhanced fluorescence AHCY inhibitor S-Adenosyl Homocysteine + AHCY Decreased fluorescence Adenosylhomocysteinase (AHCY) (EC ) or S- adenosylhomocysteine hydrolase (SAHH); is an enzyme that catalyzes the reversible hydrolysis of S-Adenosyl Homocysteine (SAH) to adenosine and homocysteine. Inhibition of AHCY results in the accumulation of SAH, a product inhibitor of S-adenosyl methionine (SAM)-dependent methyltransferases. AHCY is an important target as an antiviral and anticancer drug. Several characterized SAH inhibitors inhibit some DNA viruses (e.g. Pox viruses) and some negative stranded RNA viruses (e.g.: Marburg virus, Ebola virus & rabies). Discover more at 2

4 INTRODUCTION 2. ASSAY SUMMARY Screening compound preparation Preparation of controls Enzyme and substrate solution preparation Add enzyme solution and incubate at 37 C for 5 minutes Add substrate and incubate at 37 C for 15 minutes Stop reaction with isopropyl alcohol and incubate on ice for 5 minutes Add Thiol Detecting Reagent working solution and incubate at RT 5 minutes Read fluorescence in end point mode at Ex/Em = 392/482 nm. Discover more at 3

5 GENERAL INFORMATION 3. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance. 4. STORAGE AND STABILITY Store kit at -20ºC in the dark immediately upon receipt. Kit has a storage time of 1 year from receipt, providing components have not been reconstituted. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in Materials Supplied section. Aliquot components in working volumes before storing at the recommended temperature. 5. LIMITATIONS Assay kit intended for research use only. Not for use in diagnostic procedures. Do not use kit or components if it has exceeded the expiration date on the kit labels. Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. Discover more at 4

6 GENERAL INFORMATION 6. MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) Storage Condition (After Preparation) AHCY Assay Buffer 25 ml -20 C -20 C / +4C AHCY Reconstitution Buffer 250 µl -20 C -20 C AHCY Enzyme 1 vial -20 C -80 C Adenosine Deaminase 1 vial -20 C -80 C 3-Deazaneplanocin A (10 μm) (in DMSO) 10 µl -20 C -20 C AHCY Substrate (in DMSO) 100 µl -20 C -20 C Thiol Detecting Reagent (in DMSO) 200 µl -20 C -20 C 7. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully perform this assay: MilliQ water or other type of double distilled water (ddh 2 O) DMSO Isopropyl alcohol (chilled at -20 C) Pipettes and pipette tips Microcentrifuge Fluorescence microplate reader equipped with filter for Ex/Em = 392/482 nm 96 well plate: white plate with flat bottom Heat block or water bath Discover more at 5

7 GENERAL INFORMATION 8. TECHNICAL HINTS This kit is sold based on number of tests. A test simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions. Selected components in this kit are supplied in surplus amount to account for additional dilutions, evaporation, or instrumentation settings where higher volumes are required. Keep enzymes and heat labile components and samples on ice during the assay. Make sure all buffers and developing solutions are at room temperature before starting the experiment. Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions. Avoid foaming or bubbles when mixing or reconstituting components. Samples generating values higher than the highest standard should be further diluted in the appropriate sample dilution buffers. Ensure plates are properly sealed or covered during incubation steps. Make sure you have the appropriate type of plate for the detection method of choice. Make sure the heat block/water bath and microplate reader are switched on before starting the experiment. Discover more at 6

8 ASSAY PREPARATION 9. REAGENT PREPARATION Briefly centrifuge small vials at low speed prior to opening. 9.1 AHCY Assay Buffer: Ready to use as supplied. Equilibrate to 37 C before use. Store at 4 C or -20 C. 9.2 AHCY Reconstitution Buffer: Ready to use as supplied. Store at -20 C. 9.3 AHCY Enzyme: Reconstitute in 220 µl AHCY Reconstitution Buffer. Mix gently by pipetting up and down. Aliquot so that you have enough volume to perform the desired number of assays. Store at -80 C. Avoid repeated freeze/thaw. After reconstitution, use within two months. Keep on ice while in use. 9.4 Adenosine Deaminase: Reconstitute with 110 µl ddh 2 O. Mix gently by pipetting up and down. Aliquot so that you have enough volume to perform the desired number of assays. Store at -80 C. Avoid repeated freeze/thaw. After reconstitution, use within two months. Keep on ice while in use Deazaneplanocin A (10µM) (in DMSO): Ready to use as supplied. Warm by placing in a 37 C bath for 1 5 minutes to thaw the DMSO solution before use. NOTE: DMSO tends to be solid when stored at -20 C, even when left at room temperature, so it needs to melt for few minutes at 37 C. Aliquot inhibitor so that you have enough volume to perform the desired number of assays. Store at -20 C protected from light and moisture. Once the inhibitor is thawed, use with two months. 9.6 AHCY Substrate (in DMSO): Ready to use as supplied. Warm by placing in a 37 C bath for 1 5 minutes to thaw the DMSO solution before use. NOTE: DMSO tends to be solid when stored at -20 C, Discover more at 7

9 ASSAY PRE ASSAY PREPARATION even when left at room temperature, so it needs to melt for few minutes at 37 C. Aliquot substrate so that you have enough volume to perform the desired number of assays. Store at -20 C protected from light and moisture. Once the substrate is thawed, use with two months. 9.7 Thiol Detecting Reagent (in DMSO): Ready to use as supplied. Warm by placing in a 37 C bath for 1 5 minutes to thaw the DMSO solution before use. NOTE: DMSO tends to be solid when stored at -20 C, even when left at room temperature, so it needs to melt for few minutes at 37 C. Aliquot reagent so that you have enough volume to perform the desired number of assays. Store at -20 C protected from light and moisture. Once the reagent is thawed, use with two months. Discover more at 8

10 ASSAY PREPARATION 10.SAMPLE PREPARATION Always prepare a fresh set of samples and controls for every use Screening Compounds: Dissolve candidate inhibitors at 1000X highest final test concentration into an appropriate solvent Dilute to 4X the desired test concentration with AHCY Assay Buffer. NOTE: We suggest using different volumes of testing compounds if effective concentration is unknown. Discover more at 9

11 ASSAY PROCEDURE and DETECTION 11.ASSAY PROCEDURE and DETECTION Equilibrate all materials and prepared reagents to room temperature / 37 C prior to use. It is recommended to assay all controls and samples in duplicate Set up reaction wells: - Sample wells (S) = 25 µl test inhibitors. - Inhibitor Control wells (IC) = 1 µl 3-Deazaneplanocin A + 24 µl AHCY Assay Buffer. - Enzyme Control wells (EC) = 25 µl AHCY Assay Buffer. - Background Control wells (BC) = 25 µl AHCY Assay Buffer. NOTE: Thiol Detecting Reagent reacts with the thiol groups in the enzymes and in the homocysteine. Hence a Background Control (BC) containing AHCY and Adenosine Deaminase should be used. - OPTIONAL: Solvent control (SC) = 25 µl solvent. NOTE: preferred final solvent concentration should not be more than 2% by volume. If solvent exceeds 2%, include solvent control to test the effect on the solvent on enzyme activity Prepare AHCY Enzyme Solution: Prepare 25 µl of AHCY Enzyme Solution for each reaction: Component Enzyme Solution (µl) AHCY Assay Buffer 23 AHCY Enzyme 2 Mix enough reagents for the number of assays (samples and controls) to be performed. Prepare a master mix of the Reaction Mix to ensure consistency. We recommend the following calculation: X µl component x (Number reactions +1) 11.3 Add 25 µl of AHCY Enzyme Solution to each well. Mix gently Incubate plate at 37 C for 5 minutes AHCY Reaction Mix: Discover more at 10

12 ASSAY PRE ASSAY PROCEDURE and DETECTION Prepare 50 µl of AHCY Reaction mix for each reaction. Component Reaction Mix (µl) Background Control Mix (µl) AHCY Assay Buffer AHCY Substrate 1 0 Adenosine Deaminase 1 1 Mix enough reagents for the number of assays (samples background control) to be performed. Prepare a master mix of the Reaction Mix to ensure consistency. We recommend the following calculation: X µl component x (Number reactions +1) 11.6 Add 50 µl of Reaction Mix Solution to each of S, EC, IC and SC wells Add 50 µl of Background Control Mix to each BC well. Component The table below shows the experimental set up: Sample Well (S) (µl) 11.8 Incubate at 37 C for 15 minutes Stop the reaction by adding 50 µl of pre-chilled isopropyl alcohol into each well Mix and keep on ice for 5 min Thiol Detection Probe: Inhibitor Control (IC) (µl) Enzyme control (EC) (µl) Background control (BC) (µl) Test inhibitor compound AHCY Assay Buffer AHCY Inhibitor AHCY Enzyme Solution AHCY Enzyme Reaction Mix AHCY Background Control Mix Discover more at 11

13 ASSAY PRE ASSAY PROCEDURE and DETECTION Prepare 50 µl of Thiol Detecting Probe working solution for wells just prior to use: Component Reaction Mix (µl) DMSO 48 Thiol Detecting Probe 2 Mix enough reagents for the number of assays (samples background control) to be performed. Prepare a master mix of the Reaction Mix to ensure consistency. We recommend the following calculation: X µl component x (Number reactions +1) Add 50 µl of Thiol Detecting Reagent working solution into each well, mix and incubate at room temperature for 5 minutes. (Do not incubate more than 5 minutes.) Measure fluorescence in a microplate reader in end point mode at Ex/Em = 392/482 nm. Discover more at 12

14 DATA ANALYSIS 12.CALCULATIONS For statistical reasons, we recommend each sample should be assayed with a minimum of two replicates (duplicates) Average the duplicate reading for each test sample compound, Inhibitor Control and Enzyme control Subtract the mean fluorescence value of the Blank Control (BC) from all controls and sample readings. This is the ΔRFU Set the ΔRFU of Enzyme Control (EC) as 100% Calculate the % inhibition or % Relative Activity as follows: % Inhibition = ΔRFU of EC ΔRFU of S ΔRFU of EC 100 % Relative Activity = NOTE: ΔRFU of S ΔRFU of EC 100 If RFU of SC < RFU of EC = make a higher stock of test inhibitor, or dissolve the inhibitor in lower concentration of the solvent; or use a different solvent. If RFU of S < RFU of BC = treat as 100% inhibition and further dilute the test inhibitor and repeat the assay. Discover more at 13

15 DATA ANALYSIS 13.TYPICAL DATA Figure 1. Inhibition of AHCY Enzyme activity by 3-Deazaneplanocin A. Assay was performed following kit protocol. IC 50 (3-Deazaneplanocin A) = nm. Discover more at 14

16 RESOURCES 14.QUICK ASSAY PROCEDURE NOTE: This procedure is provided as a quick reference for experienced users. Follow the detailed procedure when performing the assay for the first time. Prepare enzyme mix, substrate mix, inhibitor (aliquot if necessary) and get equipment ready. Prepare samples and dissolve test inhibitors in suitable solvent. Prepare enzyme solution for all wells to be set up (25 µl/well) Component Enzyme Solution (µl) AHCY Assay Buffer 23 AHCY Enzyme 2 Set up plate as follows: Incubate plate at 37 C for 5 minutes.. Prior to use, prepare 50 µl AHCY Reaction Mix for each well (Number wells + 1). Component Substrate Mix (µl) Background Control Mix (µl) AHCY Assay Buffer AHCY Substrate 1 0 Adenosine Deaminase 1 1 Component S (µl) SC (µl) EC (µl) IC (µl) BC (µl) Enzyme Mix Solvent test compound Test Inhibitor Compound Assay Buffer Inhibitor control Add 50 µl of Reaction Mix Solution to each of S, EC, IC and SC wells. Discover more at 15

17 RESOURCES Add 50 µl of Background Control Mix to each BC well. Incubate at 37 C for 15 minutes. Stop the reaction by adding 50 µl of pre-chilled isopropyl alcohol (not provided) into each well. Mix and keep on ice for five minutes. Prepare 50 µl of Thiol Detecting Probe working solution for each well (Number wells + 1): Component Reaction Mix (µl) DMSO 48 Thiol Detecting Probe 2 Add 50 µl of Thiol Detecting Reagent working solution into each well Mix and incubate at room temperature for 5 minutes. (Do not incubate more than 5 minutes.). Measure fluorescence in end point mode at Ex/Em = 392/482 nm. Discover more at 16

18 RESOURCES 15.TROUBLESHOOTING Problem Cause Solution Assay not working Lower/ Higher readings in samples and Standards Standard readings do not follow a linear pattern Unanticipated results Use of ice-cold buffer Plate read at incorrect wavelength Use of a different 96- well plate Improperly thawed components Allowing reagents to sit for extended times on ice Incorrect incubation times or temperatures Pipetting errors in standard or reaction mix Air bubbles formed in well Standard stock is at incorrect concentration Measured at incorrect wavelength Samples contain interfering substances Sample readings above/ below the linear range Buffers must be at room temperature Check the wavelength and filter settings of instrument Colorimetric: Clear plates Fluorometric: black wells/clear bottom plate Thaw all components completely and mix gently before use Always thaw and prepare fresh reaction mix before use Verify correct incubation times and temperatures in protocol Avoid pipetting small volumes (< 5 µl) and prepare a master mix whenever possible Pipette gently against the wall of the tubes Always refer to dilutions on protocol Check equipment and filter setting Troubleshoot if it interferes with the kit Concentrate/ Dilute sample so it is within the linear range Discover more at 17

19 RESOURCES 16.FAQ Discover more at 18

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